Structural characterization and mechanistic elucidation of micellular aggregates present one of the most timely problems in biochemistry today. Micelles play an important role in interfacial interactions in living systems; they are also involved in lipoprotein transport and metabolic degradation, as well as membrane fusion. In order to understand the mechanisms of these processes and be able to use micelles as reaction media, their structure and properties need to be elucidated. These goals have not yet been achieved primarily because of the challenged posed by the dynamic nature of micelles. Phospholipids as water-insoluble components of all biological membranes are also manipulated in vitro in micellar aggregates. The proposed project aims at elucidation of the structural organization and dynamic behavior of phospholipids in mixed micelles. The working hypothesis is that spectroscopic studies of strategically labeled micelle- bound phospholipids with EPR-active and fluorescent reporter groups will provide useful information to advance the level of understanding of the structure and dynamics of phospholipid molecules incorporated into micellular aggregates. Employing a newly developed EPR method, for the first time bimolecular collision rates between paramagnetic probes derived from spin exchange frequencies obtained from EPR spectra will be used to determine (1) phospholipid distribution (clustered vs. interspersed), (2) location (in the core vs. at the interface), (3) orientation/confrontation (intramolecular chain-to-chain distance), and (4) dynamics (translational vs. rotational movement of spectroscopically labeled moieties). The results emerging from the new EPR method will be compared to measurements obtained using well-established fluorescence techniques (time-resolved fluorescent quenching, TRFQ, and resonance energy transfer, RET) to provide a model supported by both series of measurements. In addition, metal conformation modulation will be used to align the two fatty acid side-chain substituents parallel to each other. This will be done to delineate the micellular behavior of phospholipid molecules possessing membrane-like conformation orientation. Should the new EPR method prove to be successful in characterizing the mixed micelles here studied, it will open the possibility for applying it to other, more complicated supramolecular assemblies (e.g. phospholipid-bile salt mixed micelles) and may become a general technique for physicochemical characterization of micellular aggregates.